An intricate regulatory process governs the development of all hematopoietic lineages from precursor stem cells. A grasp of this fundamental process will help to reveal new ways of combating developmental and clonal hematological disorders such as hypoplastic anemias and leukemias. Three functional properties of stem cells are recognized; self-renewal, commitment and differentiation. The search for genes participating in these pivotal events has led to the identification of at least two classes of genes:transcriptional factors that coordinate gene expression and signal transducers. However it is far from clear that all the key classes of molecular players have been identified and it is likely that other classes of gene, as yet unrecognized, are involved in hematopoietic stem cell development. The central goal of this project is to expand the identification of novel genes which are important for development and growth of stem cells. Our proposition is that genes that are preferentially expressed in hematopoietic cells will offer a valuable focus of investigation. This proposal focuses on studying one such novel gene named GDI-D4. GDI-D4 is activated at the very early stages of hematopoietic development from totipotent stem cells. The gene maps to chromosome 12p12.3. In leukemic cases with non-random deletions involving 12p12-13, GDI-D4 resides within a region of deletion common to all cases. GDI-D4 may be a tumor suppressor. The protein functions partially as a GDP-dissociation inhibitor (GDI) of the subfamily of ras homologous proteins, rho, and is therefore one of the key regulators of the cycle of activation and inactivation of GTP binding proteins. GDI-D4 is one of very few GDIs identified so far. The specific aims of this project are to further define the cellular and biochemical function of this gene by the following steps: (1) To study the consequences of a targeted disruption of the gene in embryonal stem cells on hematopoietic cell development and function; (2) To identify and clone for protein(s) interacting with GDI-D4 protein using a double-hybrid yeast system; (3) To further investigate the tumor suppressor activity of this protein. These studies should lead to a broader appreciation of the factors regulating hematopoiesis. It may also help in the understanding of the disease mechanism of certain classes of leukemias and the identification of potential targets for therapeutic intervention. The long term objective is to widen the scope of current knowledge of the molecular determinants of hematopoietic stem cell development and to apply the knowledge in combating human diseases.